Collagenase and neutral protease1 are essential tissue dissociation enzymes (TDEs) required to release islets2 and other cells3-6 from tissue. The focus of many reports has been on the characteristics and performance of collagenase in islet isolation7,8 but a recent report by Brandhorst et al. showed that the presence of trypsin like activity (TLA) in selected lots of Serva purified collagenase when added to Clostridium histolyticum neutral protease correlated with higher human islet yields9. This post emphasizes the assays used to assess neutral protease activity present in tissue dissociation enzymes. It is the first of an ongoing series reviewing biochemical characteristics of these enzymes used in cell isolation procedures.
Historically, neutral protease assays used to assess activity in TDE products often use protein - native or derivatized casein or bovine serum albumin - instead of peptide substrates. The assay adopted early at VitaCyte was the azocasein assay which required a precipitation step with trichloroacetic acid (TCA) to separate the undigested protein from the soluble chromogenic peptides10. These assay data were problematic for several reasons: uncontrolled variability in the blank samples, low signal to noise ratio leading to high interassay coefficients of variation (typically >15%), and inconsistent performance when comparing the specific activities of thermolysin and Bacillus polymyxa protease (BP protease, i.e., Dispase®). Both enzymes have similar specificity and our earlier experience showed that thermolysin has a 2-3 fold higher specific activity than BP protease. Using azocasein this difference was minimal, causing VitaCyte to develop the fluorescent microplate assay to assess neutral protease activity.
VitaCyte’s fluorescent microplate assay used fluorescein isothyocyanate (FITC) labeled bovine serum albumin (FITC-BSA) as substrate. The same assay format was used as previously described for a collagen degradation assay11. There are several benefits for choosing a fluorescent microplate assay to assess enzyme activity. First, it is homogeneous, meaning there is no requirement to separate intact protein from released peptides. The relative fluorescence units increase during the course of proteolytic degradation reflecting the increased release of small fluorescent peptides into the assay mixture. Second, this use of a microplate enables repeated measurements of a larger number of assay samples leading to an accurate assessment of the kinetic activity of the enzyme. This also enables rapid assessment of the effect of inhibitors or other enzymes on neutral protease activity. Third, the specific activities of thermolysin and BP protease are similar to our prior experience comparing the specific activities of these purified enzymes. And lastly, the intra and interassay coefficients of variation (CVs) were <5% and <9% respectively. A comparison of results from the two assay formats are presented in the Figure below and show the difference in the specific activities between thermolysin and BP protease depending on the substrate used in the assay.
Azocasein and FITC-BSA Protease Assay Comparison
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This fluorescent microplate neutral protease assay is the only high throughput, kinetic assay used by enzyme manufacturers today for characterizing neutral protease activity as shown in the table below.
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The fluorescent microplate assay was used to assess the TLA in selected lots of purified collagenases. The sulfhydryl protease clostripain has been shown to be the enzyme primarily responsible for TLA activity. This was confirmed by strong inhibition of TLA activity by the sulfhydryl protease inhibitor N-ethylmaleimide (NEM). NEM inhibited >98% activity of a clostripain control and >96% of TLA activity in collagenase. Thermolysin and C. histolyticum neutral protease were <30% inhibited by NEM and inhibitors of other protease types (ie aspartyl-, metallo-, and serine) were ineffective at inhibiting the TLA activity (see Figure below). A previous post on VitaCyte’s website discusses the implications of the TLA in purified collagenase, suggesting that it may provide a significant contribution to the neutral protease activity in enzyme mixtures that use the Serva C. histolyticium neutral protease and collagenase.
Collagenase TLA Inhibition
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A previous report in the patent literature indicated that a synergistic increase in activity assays was observed when purified collagenase, neutral protease and clostripain were mixed. C. histolyticum neutral protease is similar to the specificities of thermolysin and Dispase® which cleave on the amide side of hydrophobic amino acid such as leucine, whereas clostripain targets primarily arginine. Enzymes of different specificities work cooperatively to accelerate the proteolysis of a protein substrate. These data are summarized in a prior post on VitaCyte’s website. This has significant implications for understanding the optimal protease blends and suggests that definition of key enzyme components (both collagenase and neutral proteases) will be essential to prepare TDE blends that perform more consistently in cell isolation.
While the exact role of the neutral protease is still not completely understood because of the complexity of the natural substrate in the extracellular matrix that is composed of collagen, hyaluronic acid, proteoglycans, fibronectin, laminin and elastin. All current assays use surrogate substrates that provide insight on the characteristics of proteases and how to prepare them more consistently. The FITC-BSA assay described above is a suboptimal surrgate assay for understanding neutral protease activity required for cell isolation. However, it is an improvement over other traditional assays and serves as a step to prepare more complex substrates that will provide a more realistic model of the protease’s role in tissue dissociation.
References:
1. Neutral protease: a proteolytic enzyme active at neutral pH.
2. McShane P., Sutton R., Gray D.W., and Morris P.J. (1989) Protease activity in pancreatic islet isolation by enzymatic digestion. Diabetes. 38 Suppl 1, 126-128.
3. Kono T. (1969) Role of collagenases and other proteolytic enzymes in the dispersal of animal tissues. Biochimica Biophysica Acta 178, 397-400.
4. Hatton M.W., Berry L.R., Krestynski F., Sweeney G.D., and Regoeczi E. (1983) The role of proteolytic enzymes derived from crude bacterial collagenase in the liberation of hepatocytes from rat liver. Identification of two cell-liberating mechanisms. Eur J Biochem 137, 311-318.
5. Hefley T.J., Stern P.H., and Brand J.S. (1983) Enzymatic isolation of cells from neonatal calvaria using two purified enzymes from Clostridium histolyticum. Experimental Cell Research 149[1], 227-236.
6. Suggs W., Van Wart H., and Sharefkin J.B. (1992) Enzymatic harvesting of adult human saphenous vein endothelial cells: use of a chemically defined combination of two purified enzymes to attain viable cell yields equal to those attained by crude bacterial collagenase preparations. Journal of Vascular Surgery 15, 205-213.
7. Barnett M.J., Zhai X., LeGatt D.F., Cheng S.B., Shapiro A.M.J., and Lakey J.R.T. (2005) Quantitative assessment of collagenase blends for human islet isolation. Transplantation 80, 723-728.
8. Kin T., Zhai X., O’Gorman D., and Shapiro A.M. (2008) Detrimental effect of excessive collagenase class II on human islet isolation outcome. Transplant International 21[11], 1059-1065
9. Brandhorst H. , Friberg A., Andersson H.H., Felldin M., Foss A., Salmela K., Lundgren T., Tibell A., Tufveson G., Korsgren O., and Brandhorst D. (2009) The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation. Transplantation 87, 370-375. 2009.
10. Sarath G., De La Motte R., and Wagner F.W. (1989) Protease assay methods. In Proteolytic Enzymes, A Practical Approach, pp. 25-55. IRL Press, New York.
11. McCarthy R.C., Spurlin B., Wright M.J., Breite A.G., Sturdevant L.K., Dwulet C.S., and Dwulet F.E. (2008) Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation. Transplantation Proceedings 40[2], 339-342.
12. Twining S.S. (1984) Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Analytical Biochemistry 143, 30-34.
13. Matsubara H. (1970) Thermolysin. Methods Enzymology 19, 642-650.
14. Y Lin, GE Means, and RE Feeney (1969) The action of proteolytic enzymes on N,N-dimethyl proteins. Journal Biological Chemistry 244, 789-793.
15. Voss E.W., Workman C.J., and Mummert M.E. (1996) Detection of protease activity using fluorescence-enhancement globular substrate. BioTechniques 20, 286-291.
16. Sigma neutral protease assay : http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/p1512enz.Par.0001.File.tmp/p1512enz.pdf.
17. Worthington neutral protease assay: http://www.worthington-biochem.com/DISP/assay.html.
Categories: Neutral Protease
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